The overall goal of this grant application is to understand the regulatory pathways that control the regenerative capacity of hematopoietic stem cells (HSCs) during stress hematopoiesis. A clear understanding of how signaling pathways are used to balance HSC self-renewal and differentiation for efficient hematopoietic regeneration is still lacking. It is unclear how signaling pathways control HSC self-renewal in concert with other regulatory elements. This lack of knowledge has hampered our ability to prevent the decline in HSC regenerative capacity associated with stress hematopoiesis and, consequently, has limited the success of HSC-based therapies that require high numbers of HSC. We have recently discovered a novel and clinically important regulatory network of HSC self-renewal, involving crosstalk between Rho GTPase signaling pathways and mitochondria functions that limit HSC regenerative capacity. We reported that genetic deletion of p190-B Rho GTPase Activating Protein (p190-B RhoGAP [p190-B]); a suppressor of Rho GTPase activity, in mice enhanced long-term HSC engraftment and prevented HSC depletion over serial competitive repopulation. P190-B knock-down in human CD34+ cells preserved huCD34+ functions during ex vivo culture. Single cell assays revealed that p190-B loss promoted HSC self-renewal decision over differentiation during divisions; but HSC quiescence and blood lineage development were not affected. Mechanistically, p190-B loss enhanced HSC self-renewal by limiting mitochondrial oxidative stress and subsequent abnormal activation of an autocrine TGF/p38MAPK stress signaling pathway. We propose that p190-B uses mitochondria to convert oxidative stress into autocrine cytokine signals to instruct HSC fate decision during HSC regeneration. Aim 1 will determine mechanism linking p190-B and Rho signaling to autocrine TGF1 pathway for HSC self-renewal via. Aim 2 will define how p190-B uses mitochondria and oxidative energy to modulate TGF - mediated HSC self-renewal. Aim3 will test the effects of pharmacological inhibition of these pathways on human CD34+ fitness in xeno-transplant models. The proposed studies are innovative because it explores the role of major stress pathways in an underexplored fundamental aspect of HSC biology - i.e. a HSC decision to self-renew or to differentiate independent on mature lineage differentiation or HSC quiescence. The work is expected to yield novel insights in mechanism of HSC self-renewal by crosstalk between signaling and mitochondrial metabolism. This study may ultimately lead to the identification of novel targets for pharmacological intervention in regenerative medicine.